The transcriptional and DNA binding activity of peroxisome proliferator-activated receptor (cid:1) is inhibited by ethanol metabolism: a novel mechanism for the development of ethanol-induced fatty liver

Fatty acids are ligands for the peroxisome proliferator activated receptor (cid:1) (PPAR (cid:1) ). Fatty acid levels are increased in liver during the metabolism of ethanol and might be expected to activate PPAR (cid:1) . However, ethanol inhibited PPAR (cid:1) activation of a reporter gene in H4IIEC3 hepatoma cells expressing alcohol-metabolizing enzymes, but not in CV-1 cells which lack these enzymes. Ethanol also reduced the ability of the PPAR (cid:1) ligand WY14643 to activate reporter constructs in the hepatoma cells or cultured rat hepatocytes. This effect of ethanol was abolished by the alcohol dehydrogenase inhibitor 4-methylpyrazole and augmented by the aldehyde dehydrogenase inhibitor cyanamide, indicating that acetaldehyde was responsible for the action of ethanol. PPAR (cid:1) /retinoid X receptor (RXR) extracted from hepatoma cells exposed to ethanol or acetaldehyde bound poorly to an oligonucleotide containing peroxisome proliferator response elements. This effect was also blocked by 4-methylpyrazole and augmented by cyanamide. Furthermore, in vitro translated PPAR (cid:1) exposed to acetaldehyde failed to bind DNA . Thus, ethanol metabolism blocks transcriptional activation by PPAR (cid:1) , in part due to impairment of its ability to bind DNA. This effect of ethanol may promote the development of alcoholic fatty liver and other hepatic consequences of alcohol abuse.


Introduction
The liver coordinates synthesis of fatty acids, esterification of triacylglycerols, and their packaging into very low density lipoproteins (VLDL) for export during fed conditions, while in fasting it controls the rates of -oxidation and ketogenesis. By balancing these processes, the liver handles large amounts of fat without accumulating triacylglycerol. Many homeostatic responses of the hepatocyte to FFA are modulated by peroxisome proliferator-activated receptor (PPAR). FFA are endogenous ligands for PPAR (1)(2)(3)(4) and numerous genes involved in fat metabolism contain peroxisome proliferator response elements (PPREs) in their promoters. It has been suggested that this constitutes a feed back control system: elevated FFA activate PPAR, inducing a battery of enzymes (peroxisomal -oxidation, mitochondrial -oxidation, and microsomal fatty acid hydroxylation (which initiates 7-oxidation)) involved in FFA oxidation (5,6), which serve to reduce the level of FFA. However, in certain forms of liver disease, this fine balance is disrupted, and elevated levels of hepatocellular free fatty acids (FFA) and triacylglycerol occur.
The most common liver disease in which fatty acid metabolism is deranged is alcoholic liver disease. Alcohol metabolism alters the intramitochondrial redox potential via generation of NADH by alcohol dehydrogenase (ADH). This impairs -oxidation and tricarboxylic acid cycle activity (7), resulting in elevated FFA, increased formation of triacylglycerol, and increased rates of VLDL synthesis and secretion (8,9). Paradoxically, the fatty liver persists despite attenuation of the altered redox state after chronic ethanol administration (10). Fatty liver is not necessarily by guest on March 20, 2020 http://www.jbc.org/ Downloaded from Andrea Galli et al. 4 benign: the development of liver injury in a rat model is clearly dependent upon the amount and type of fat in the diet (11,12) and a disproportionate elevation of liver FFA after ethanol administration may contribute to the susceptibility of women to alcoholic liver disease (13).
Genetically obese mice and rats with hepatic steatosis are unusually sensitive to the effects of endotoxin (14). Furthermore, a number of other compounds, including valproic acid, amiodarone, and perhexilene, are postulated to cause liver injury by way of inhibition ofoxidation (15,16). Thus, several lines of evidence suggest that liver injury may occur when fatty acid oxidation or esterification and export are inadequate.
One would predict that ethanol consumption would induce the PPAR battery of proteins by elevating intracellular fatty acid levels. Although alcohol consumption resulted in peroxisomal proliferation in humans (17) and alcohol feeding of rats induced cytochrome P450 4A1 (lauryl 7-hydroxylase, (13)) and liver fatty acid binding protein (18), other typical responses to peroxisome proliferators were impaired by ethanol. The excretion of dicarboxylic fatty acids was increased in alcohol-fed animals (13), due to increased lauryl hydroxylase activity but failure of induction of acyl-CoA oxidase (19). Medium chain acyl-CoA dehydrogenase activity, the gene for which has a PPRE in its proximal promoter (20), was reported to be decreased by ethanol feeding (21). Thus, chronic ethanol feeding apparently does not activate a full PPAR response. One group has reported that PPAR mRNA was decreased in the livers of rats chronically fed alcohol via gastric lavage, and that several PPAR-inducible enzymes were not increased in these animals (22). It is noteworthy that fatty liver and steatohepatitis, hallmarks of alcoholic liver injury, are also observed in both PPAR (23) and acyl-CoA oxidase (24) (25)), and the expression plasmids for murine PPAR, , and γ were the kind gifts of Dr. Ronald Evans, Salk Institute (26). pALDH3'-BLCAT was used as an HNF-4 responsive reporter and was previously described (27). Expression plasmids for apolipoprotein regulatory protein 1 (ARP-1) and chicken ovalbumin upstream promoter transcription factor (COUP-TF) were from Dr. H. Nakshatri (Indiana University) and that for hepatocyte nuclear factor 4 (HNF-4) was from Dr. Frances Sladek (University of California, Irvine).
Transfection of tissue-culture cells All cells were grown in modified Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 g/ml streptomycin, and 63 g/ml penicillin G. The day before transfection, the cells were plated at 10 6 cells/100 mm dish. For studies on PPAR α and γ, the cells were transfected with 10 g of reporter plasmid (PPRE 3 -tk-luciferase), 20 g of receptor expression plasmid, and 5 g of pSV 2 CAT (as an internal control for transfection efficiency) by calcium phosphate by guest on March 20, 2020 http://www.jbc.org/ Downloaded from Andrea Galli et al. 7 precipitation (28). For studies on HNF4, the reporter contained four copies of an HNF-4 response element from the aldehyde dehydrogenase 2 promoter cloned in pBL2CAT (28) and the internal control was SV40-luciferase. For studies on ARP-1 and COUP-TF, the reporter was SV40luciferase (which is activated by these two orphan receptors (29)) and because of problems with effects of these receptors on other promoters, the activity was expressed per µg cell extract protein.
Four hours later the cells were exposed to PBS containing 15% glycerol for 3 min. The cells were rinsed twice with PBS and fresh MEM with 10% charcoal-stripped fetal bovine serum was added.
Twenty-four to forty-eight hours after transfection, cells were washed twice with PBS and lysed in 150 l of a buffer containing 25 mM Tris, pH 7.8, 2 mM EDTA, 20 mM DTT, 10% glycerol, and 1% Triton X-100. Fifty l of cell extract was incubated with luciferase assay reagent based on the original protocol of deWet (30). CAT activity was measured as described previously (31). The conversion of chloramphenicol to its acetylated products was quantified on an AMBIS -scanner.
Primary hepatocyte suspensions were isolated from male Sprague-Dawley rats (Harlan Laboratory Animals for Research, Indianapolis, IN) as previously described (32,33). Briefly, rats were anesthetized with pentobarbital (50-100 mg/kg body weight), their portal veins were cannulated with a 16-gauge catheter, and the livers were perfused with Ca ++ , Mg ++ -free Hanks' A solution, followed by Hanks' B solution containing Ca ++ , Mg ++ , and 0.05% collagenase dexamethasone and thyroxine, 1 nM insulin, and 10% FBS. They were transfected 4 hours after plating by calcium phosphate precipitation according to the method of Ginot (34). Twenty four hours later the cells were treated 100 M WY14,643 for an additional 24 hr before harvesting the cells for assay of reporter enzymes as described above.

Isolation of nuclear protein extracts
Nuclear proteins were isolated from cultured cells based on a micropreparation method (35).
In vitro synthesis of receptor proteins PPARs and RXR were synthesized using a rabbit reticulocyte lysate system (Promega in vitro transcription/translation kit). The production of protein of the expected molecular weight was monitored by labeling with 35  with 1-2 µg of non-specific competitor DNA (poly (dIC)) in binding buffer containing 10 mM Hepes, pH 7.9, 60 mM KCl, 1 mM EDTA, and 7% (v/v) glycerol on ice for 15 min. Where indicated, specific competitor oligonucleotides were added before the addition of labeled probe and incubated for 15 min on ice. For supershift assays, antibodies were added and the mixture was incubated an additional 1-2 hours. Labeled probe (20,000 cpm) was added last and the reaction incubated an additional 15 min on ice. Reaction mixtures were electrophoresed on a non-denaturing 4% acrylamide gel and subjected to autoradiography. Anti-PPAR antibody was from Santa Cruz Biotechnology and anti-RXR was donated by Drs. C. Rochette-Egly and P. Chambon.

Western blotting
Nuclear extracts (20 µg protein) were fractionated in an 8% SDS-PAGE gel and electroblotted to nitrocellulose filters. PPAR was visualized using antiserum from Santa Cruz Biotechnology. Detection of the protein bands was performed using the Amersham ECL kit.

Effects of ethanol on transcriptional activation by PPAR in cells with and without the enzymatic capacity for ethanol oxidation.
The activity of a PPAR-responsive reporter gene (PPRE 3 -tk-luciferase, containing 3 copies of the PPRE from the acyl-CoA oxidase gene) was used as an index of PPAR function in CV-1 and H4IIEC3 hepatoma cells with or without co-transfected PPAR (Table I). These cells contain low amounts of PPAR protein on Western blots ( Figure 2B, lane 2) but both cell lines contained immunoreactive retinoid X receptor (RXR, (36)), the required dimerization partner for PPAR (25). An important difference between CV-1 and H4IIEC3 cells was the presence of enzymes capable of oxidizing ethanol in the latter cells (37,38). Responses of the reporter were relatively small (no more than two-fold induction) in the absence of co-transfected PPAR.
Clofibrate markedly induced the reporter activity in CV-1 cells transfected with PPARα. In the hepatoma cells, the reporter activity was much less dependent on the presence of clofibrate (36).
Ethanol at a physiologically relevant concentration of 20 mM inhibited clofibrate-independent and -stimulated activity of PPRE 3 -tk-luciferase by PPAR by over 50% in the hepatoma cells (Table I). Ethanol had no effect on basal or clofibrate-stimulated PPAR action in the CV-1 cells, either in the presence or absence of transfected PPAR.
The effect of ethanol on the ability of the more potent and specific PPARα agonist WY14,643 was also tested. In duplicate experiments with H4IIEC3 cells transfected with the PPARα expression plasmid and reporter, WY14, 643 at 100 µM increased reporter activity by 591 ± 49 %. Ethanol (20 mM) reduced the basal activity of the reporter to 45 ± 4%, and decreased the WY14,643-stimulated activity to 216 ± 134% of the control level (means ± standard error). We also tested the effect of WY14,643 on primary hepatocyte cultures that were transfected with the PPAR reporter plasmid to see if ethanol also inhibited the activity of the endogenous rat PPARα. WY14,643 stimulated reporter activity by 433 ± 107. Ethanol (20 mM) reduced basal activity to 57 ± 3 % and WY14,643-stimulated activity to 128 ± 32% of control levels (means ± standard errors for four replicate experiments). Thus, ethanol reduced the activity of the reporter by about 50% in both the basal and WY-14,643-stimulated cells, similar to the magnitude of the effect on clofibrate-stimulated activity. Further, the effect was also seen in primary cultures of hepatocytes, indicating that the rat PPARα is also sensitive to ethanol.
To determine if this effect of ethanol was restricted to PPARα, additional transfection assays were performed using PPARγ, HNF4, ARP-1, or COUP-TF. These receptors are structurally related to PPARα and each recognizes DR-1 promoter elements. Compared with hepatoma cells transfected with PPARγ alone (n= 5, means standard errors), clofibrate increased activity of PPRE 3 -tk-luciferase by 189 20 %, while ethanol reduced activity to 60 3 %, and reduced the clofibrate-stimulated activity to 154 21%. These small differences were statistically significant. Transfection of the H4IIEC3 cells with an HNF-4 expression plasmid stimulated its reporter plasmid expression (pALDH3'-BLCAT containing 4 copies of an HNF-4 response element from the aldehyde dehydrogenase promoter) by 956 159% in the absence and 1034 109% in the presence of ethanol (n= 3, not significant). ARP-1 stimulated its reporter plasmid expression (SV40-luciferase (29)) by 8222 1776% in the absence and 12203 6875% in the presence ethanol (n = 4, not significant). COUP-TF stimulated its reporter plasmid expression (SV40-luciferase) by 10,408 1092% in the absence and 9312 3432% in the presence of ethanol (not significant, n = 4). The large errors observed in the transfections with ARP-1 and COUP-TF were related to the use of cellular protein for normalizing the data, rather than an internal control plasmid (29). Thus, the effect of ethanol was relatively specific for PPARα, although the small effect on the γ isoform was studied further with in vitro translated receptor (below).

Effects of inhibitors of ethanol metabolism and acetaldehyde on PPAR function
The ADH inhibitor 4-methylpyrazole and the aldehyde dehydrogenase (ALDH) inhibitor cyanamide were then used to determine if the effect of ethanol on PPAR was dependent on its metabolism (Table II). Neither compound affected PPRE 3 -tk-luciferase activity in the hepatoma cells in control experiments. However, 4-methylpyrazole completely prevented the effect of ethanol on PPAR function, while cyanamide augmented the effect. This suggested that acetaldehyde generated from ethanol was responsible for the inhibition of PPAR action, and indeed, low levels of exogenous acetaldehyde (50-150 M) inhibited PPRE 3 -tk-luciferase reporter activity, both in H4IIEC3 cells and CV-1 cells (Table III) Table I; means standard error for 3 replications), indicating that acetaldehyde-protein adduct formation may explain the inhibitory effect of ethanol metabolism.

Effect of ethanol and acetaldehyde on DNA binding ability of PPAR
To understand how ethanol metabolism impaired PPAR function, it was of interest to study the effect of ethanol on the ability of nuclear factors to bind the PPRE in EMSA. In preliminary experiments, ethanol was found to have no effect on the level of endogenous RXR in the hepatoma cells. Nuclear extracts from untransfected H4IIEC3 cells contained proteins that retarded the mobility of the PPRE oligonucleotide ( Figure 1). The major and minor bands appeared to be specific, in that they were competed with unlabeled oligonucleotide (lanes 3-5).
These bands were not PPARα, since the hepatoma cells contain very low levels of PPARα ( Figure 2B, lane 2) and the bands could not be shifted with antibody to PPARα (not shown). The bands likely represent other nuclear factors present in H4IIEC3 cells that can bind to DR-1 elements, such as HNF-4. When the cells were exposed to ethanol, alone or in the presence of inhibitors of its metabolism, there was no change in the intensity of the bands.
We then analyzed nuclear extracts from H4IIEC3 cells that were transfected with the by guest on March 20, 2020 http://www.jbc.org/ Downloaded from PPARα expression plasmid (Figure 2A and B). The intensity of the major band was markedly increased in the transfected cells, which contained large amounts of PPARα seen by western blotting (Figure 2A and B, lane 3), and there was a prominent shift induced with anti-PPARα.
Binding was again competed with unlabeled competitor oligonucleotides (lanes 4-6). The major band could also be shifted with antibody to RXR (not shown). The more slowly migrating band was also more intense in the transfected cells. The identity of this band is uncertain; however, it might represent nuclear receptors bound to other factors such as NRBF-1 (43) or PBP165 (44).  (45)) reduced the ability of extracted PPAR to bind DNA ( Figure 3A). This treatment of the cells did not reduce the amount of immunoreactive PPAR present in the nuclear extracts ( Figure 3B). Acetaldehyde also reduced the intensity of the more slowly moving band. Inclusion of pyridoxal phosphate in the culture medium prevented the inhibitory effects of ethanol and acetaldehyde on DNA binding activity (not shown).
To further document that the nuclear factor binding that was reduced by ethanol and acetaldehyde was PPARα, antibody against this factor was used to super-shift the binding complex ( Figure 4). This autoradiogram was exposed for a shorter time than Thus, the ethanol, by way of acetaldehyde, dramatically reduces the ability of PPARα to bind to DNA.

Effect of acetaldehyde on DNA binding ability of in vitro translated PPARs
Because there is evidence that acetaldehyde can alter protein function via covalent modification, the effect of acetaldehyde on in vitro synthesized PPAR and RXR was examined ( Figure 5A Because there was a modest reduction in the ability of PPARγ to activate the reporter in transfection studies, we also studied the effect of acetaldehyde on the ability of the PPARγ and δ isoforms to bind DNA ( Figure 5B). This experiment was carried out as described for PPARα ( Figure 5A). The preformed heterodimers (lanes 10 and 11) appeared to somewhat more stable than the receptor subunits incubated alone (lanes 8 and 9), as was seen with PPARα. Treatment of the RXR with acetaldehyde did not dramatically reduce its ability to form a DNA-binding complex with either PPARγ or δ (lanes 14 and 15). As with PPARα, both PPARγ and δ were sensitive to pre-incubation with acetaldehyde (lanes 16 and 17). The preformed heterodimers appeared to be somewhat more resistant to the effect of acetaldehyde than the corresponding PPARα/RXR complex (lanes 19 and 20). However, additional studies are needed to determine if the intracellular and in vitro effects of acetaldehyde on PPAR function involve similar mechanisms. It will also be important to examine the ability of other biologically occurring aldehydes (e.g., aldehydic products of lipid peroxidation or glucose) to affect PPAR function. We also observed sensitivity of PPARγ and δ to acetaldehyde in vitro, although the kinetics of inactivation were not formally studied to allow quantitative comparisons of the PPAR isoforms.
Earlier work has shown that exposure to ethanol increases the level of fatty acids in hepatocytes (8,9). The results of the present work show that ethanol also can impair the function of PPAR. The failure of induction of PPARα-controlled genes such as those for peroxisomal β-oxidation and medium chain acyl-CoA dehydrogenase could thus contribute to the development of alcoholic fatty liver. This effect of ethanol may be responsible for the persistence of fatty liver despite a return of the redox state toward normal during chronic ethanol administration (10). Inhibition of PPAR function may also contribute to more serious alcoholic hepatic injury. Indeed, this suggests that pharmacologic or nutritional maneuvers that activate the PPAR system may ameliorate the hepatotoxicity of ethanol. However, ethanol feeding does not uniformly inhibit expression of genes known to contain PPREs (13,18). This The actions of ethanol on PPARγ also deserve additional study. We have shown that PPARγ may play an important role in the control of proliferation of hepatic stellate cells (53).
The expression of PPARγ decreases as the cells proliferate after being plated on plastic substrate, and activated PPARγ antagonizes the actions of platelet-derived growth factor, a major contributor to proliferation of stellate cells. We hypothesize that the high intra-hepatic levels of acetaldehyde occurring during prolonged alcohol consumption inhibits PPARγ and renders the stellate cells more susceptible to activation. This could contribute to the pathogenesis of alcoholic cirrhosis as well as the increased risk of hepatic fibrosis in patients with hepatitis C who drink heavily. Further, PPARγ is extremely important for the differentiation of preadipocytes to adipocytes, in the control of sensitivity to the actions of insulin, and in the pathogenesis of atherosclerosis (54,55). Although the multiple roles of this PPAR isoform is still incompletely understood, inhibition of PPARγ function by heavy ethanol consumption might contribute to insulin resistance, syndrome X, and accelerated cardiovascular disease. This possibility deserves further study. The cells were transfected as described in Table I with the PPAR expression plasmid, internal control plasmid, and the PPRE 3 -tk-luciferase reporter; the inhibitors were added 24 hours later and the cells were exposed to ethanol (20 mM) where indicated beginning at the same time. The cells were harvested for assay of the reporter enzymes at 48 hours; data are reported as in      In the lanes 4,6, 8, 10, and 12, antibody to PPARα was added to the binding reaction prior to addition of radiolabeled DNA and incubated for 1-2 hours prior to electrophoresis. With this shorter autoradiographic exposure, the major band is resolved to 2 bands, both of which are shifted by anti-PPAR antibody.